Microorganisms having proliferation properties have recently been detected from acidic foods and beverages, particularly acidic beverage such as raw fruit juice and fruit juice, resulting in a problem. In general, bacteria hardly generate in an acid range like fruit juice and fruit juice beverage. However, thermotolerant acidophilic bacteria having thermotolerant spores among acidophilic bacteria which is fond of the acid range hardly die out under the conditions of a sterilization temperature of the beverage (usually about 93° C.), thus constituting a large problem. Typical thermotolerant acidophilic bacteria include bacteria of the genus Alicyclobacillus. It has been reported that two species (A. acidoterrestris and A. acidiphilus) among bacteria of the genus Alicyclobacillus contaminate fruit juice and fruit juice beverage to cause proliferation. Also these bacteria constitute a large problem on quality because a nasty smell component, guaiacol, is produced during proliferation. Particularly, A. acidoterrestris is isolated from fruit juice with high frequency and is considered as an indicator bacterium in control of microorganism of fruit juice and fruit juice-containing beverage.
As a method for detecting such bacteria, there have been developed, e.g., a culturing method and a method for detecting a specific gene by PCR, which are described in Patent Documents described hereinafter. However, the culturing method, which requires no expensive devise, require the culture or detection time of about 48 hours or more after pre-culture and a simple method for detection within a short time has never been known.
Japanese Patent No. 3177367 discloses a method for detecting acidophilic spore-forming bacteria using an acid culture medium containing aldehyde and a detection kit. It also discloses that, since acidophilic spore-forming bacteria converts vanillin into guaiacol and guaiacol emits a strong odor, the presence of acidophilic spore-forming bacteria in a test sample can be detected within a short time without confirming the formation of colony by adding a test sample such as fruit juice in a vanillin-containing medium.
Japanese Patent Laid-Open Publication No. 8-140696 discloses, in a method for detecting thermotolerant acidophilic bacteria having proliferation properties in acid fruit juice or acidic beverage, the presence of the bacteria can be evaluated by the presence or absence of ω-cyclohexane fatty acid using a GC-MS method and the like.
Japanese Patent Laid-Open Publication No. 10-234376 discloses a method for detecting thermotolerant acidophilic bacteria having proliferation properties in acid fruit juice or acidic beverage, which comprises detecting and identifying microorganisms belonging to the genus Alicyclobacillus by subjecting a nucleic acid coding for an enzyme involving in bio-synthesis of ω-cyclohexane fatty acid to the PCR reaction using a nucleic acid primer having a specific base sequence.
Although various dairy products with vanilla flavor are commercially available, the production of a nasty smell component, guaiacol, associated with proliferation of specific Bacillus may cause deterioration of flavor, if the conditions are satisfied. However, harmfulness of microorganisms belonging to the genus Bacillus has little been noticed, and thus a method for detecting harmful microorganisms has scarcely been reported.
The present inventors have hitherto carried out a challenge test (indicator bacteria proliferation test method) using Alicyclobacillus acidoterrestris as a means for understanding proliferation properties of bacteria in a specimen. In this test method, bactericidal conditions are determined by inoculating 101−3/ml of spores (or vegetative cells) into a specimen such as beverage, standing at 35° C. for 2 to 3 weeks, measuring an increase or decrease of bacteria with a lapse of time using a plating culture method (requiring additional 3 to 5 days), and performing risk analysis due to the thermotolerant acidophilic bacteria in the beverage. Therefore, the determination requires at least a half-month.